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Keywords:

  • development;
  • Doppel;
  • goat;
  • ovaries;
  • prion;
  • testis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Expression of the goat prion protein gene locus was assessed by reverse transcriptase-polymerase chain reaction on testes and ovaries at various developmental stages. A weak and stochastic expression of the PRNP and PRNT genes was observed. For PRNT, it is consistent with the detected deletions of two single nucleotides within its open reading frame in ruminant genes. PRND was expressed in both tissues at all stages. Whereas its expression is constant in the ovaries, it increases in testes between 36 and 46 days postcoitum (dpc) and remains high thereafter. In testes, Doppel was found in the nucleus of germinal cells and in the cytoplasm of Leydig cells at 44 dpc. It was detected in the cytoplasm of Leydig cells and of some Sertoli and germinal cells at 62 dpc. In the ovaries, it was observed in the nucleus of germinal cells at 44 dpc and mainly in their cytoplasm at 62 dpc. This expression pattern was shown to parallel that of C-kit and suggests Doppel involvement in early testis differentiation. Developmental Dynamics 236:836–842, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The human prion protein gene locus comprises three identified genes: the PRNP gene that encodes for the PrPc protein, the PRND gene encoding the Doppel protein, and a newly identified gene called PRNT (Makrinou et al., 2002). PrPc is a glycophosphatidylinositol-anchored glycoprotein mainly located in the membrane of many cells. Its function remains poorly understood. An abnormal isoform of this protein, called PrPsc, is suspected to be the infectious agent responsible for prion diseases (for reviews, see Prusiner, 1982; Weissmann, 1999; Johnson, 2005). PRNP is expressed in most of the tissues analyzed so far with the highest amounts present in the brain. In mice and ram, Prnp was found to be expressed in germ cells (Shaked et al., 1999; Weber et al., 2003; Ecroyd et al., 2004), but its invalidation in mice does not induce a fertility-associated phenotype (Bueler et al., 1992).

Doppel is also a glycophosphatidylinositol-anchored glycoprotein that shares similar structures with PrPc (Mo et al., 2001). The PRND gene has been characterized in several species, including human, rat, mice, sheep, and cows (Moore et al., 1999; Tranulis et al., 2001; Essalmani et al., 2002). This gene is located several kb downstream of PRNP, in a similar orientation. It was found to be highly expressed in testis and to a lesser extent in several other tissues such as ovaries and spleen. Some species differences in its expression pattern were reported (Tranulis et al., 2001). Similarly, recent immunohistochemical studies performed in human, mouse, rat, boar, ovine, and bovine reproductive tracts revealed both developmental and spatial differences in Doppel expression profiles (Peoc'h et al., 2002; Rondena et al., 2005; Uboldi et al., 2005; Espenes et al., 2006; Serres et al., 2006). Prnd knockout in mice resulted in male sterility associated with a sperm inability to perform the acrosome reaction and with elevated levels of oxidative DNA damage (Behrens et al., 2002; Paisley et al., 2004). It was also observed that, in the absence of PrP, ectopic expression of Doppel induced the degeneration of Purkinje cells (Mo et al., 2001; Rossi et al., 2001; Anderson et al., 2004; Al Bersaoui et al., 2005). Despite these observations, the function of the Doppel protein remains unknown.

In human, a novel gene was identified 3 kb 3′ to PRND (Makrinou et al., 2002). This gene, called PRNT, is specifically expressed in adult testis. It encodes for three alternatively spliced transcripts that share a putative open reading frame (ORF) of 94 amino acids (aa) in length. Although PRNT shows homology to expressed sequence tags of various species (Makrinou et al., 2002), it has only been characterized in humans. In bovine, a putative PRNT gene has been identified in silico that encodes for an N-terminally truncated protein homologous to its human counterpart and that is located 6 kb 3′ to PRND (GenBank gi 78771396). However, other studies conclude that PRNT is only present in primates and not in bovine (Choi et al., 2006). To gain further insight of the structure and expression profile of the prion protein gene locus in ruminants, we have searched for PRNT-related sequences in goat genomic DNA and analyzed the expression pattern of the three genes in goat testes and ovaries at various critical developmental stages.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Goat Genomic DNA Contains Sequences Highly Homologous to the Putative Bovine PRNT, Reminiscent of Human PRNT

A putative bovine PRNT gene was recently identified (GenBank gi:78771396). Compared with its human counterpart, this gene encodes for an N-terminally truncated homologous protein of 55 aa in length (Fig. 1). This size difference results from two single nucleotide deletions in the 5′ sequence of the bovine region homologous to the human PRNT ORF that induces the occurrence of a premature stop codon. However, the translation process can be theoretically reinitiated at a downstream-located AUG initiation codon (Fig. 1). Sequencing analysis of polymerase chain reaction (PCR) -amplified genomic fragments encompassing the putative PRNT ORF allowed us to confirm the published bovine sequence (GenBank gi:78771396) and to reveal that the goat genome contains a 100% 227-bp-long homologous region (EMBL: AM412782). The occurrence of a premature stop codon in the bovine or goat ORF induces by two single nucleotide deletions would not necessarily be detrimental because (1) a second AUG initiation codon is located downstream; (2) the upORF will only be a few codons long (21, see Fig. 1); and (3) in the ruminant sequence, upstream of the second AUG, a G is located in position +4 (Kozak, 1997, 2005). Although the human and ruminant PRNT-encoded proteins share 55% identities, at the nucleotide level, the homology between the two sequences is too low to be detected by BLAST (data not shown). This observation might explain the recently published lack of detection of PRNT in the bovine genomic sequence (Choi et al., 2006). Our current data demonstrate the occurrence of PRNT-related sequences in ruminant genomes. In humans, the three identified genes of the prion protein gene locus are all expressed in testis. We next investigated if this finding was also the case in goats.

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Figure 1. Sequence comparison of the human, bovine, and goat PRNT ORF. Top lane Human: Human gene sequence (nucleotide [nt] 933-1275 in GenBank gi: 31343531). Second lane Bovine: Bovine gene sequence (nt 4064-4405 in GenBank Locus DQ205538). Third lane Goat: Determined goat gene sequence (EMBL: AM412782). Human (in red): human PRNT amino acid (aa) sequence. Bovine or Bov-Gt (in red): Bovine or bovine and goat PRNT aa sequence, respectively. -, identical nucleotide; *, nucleotide deletion; $, stop codon. Identical aa between the human and the bovine proteins are in boldface type.

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Only PRND Appears to Be Regulatory Expressed in Goat Testes and Ovaries

Expression of the three genes was assessed by reverse transcriptase-PCR (RT-PCR) analyses performed on total RNA extracted from goat testes and ovaries at various developmental stages from six animals (Fig. 2). Despite the use of a high number (40) of amplification cycles, weak and inconsistent signals were obtained for PRNP and PRNT at all the stages studied on RNA samples derived from six different fetuses and between repeated experiments. This result strongly suggests that these two genes are very weakly and stochastically expressed in these two tissues. This observation is consistent with previously reported data showing that, in the reproductive tract of the ram, PrP is mainly observed in the epididymis and only very weakly in the testis (Gatti et al., 2002; Ecroyd et al., 2004). For PRNT, it suggests either that the expression pattern of this gene differs between ruminant and human or, most probably, that ruminant PRNT is a pseudogene.

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Figure 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis. RT-PCR were performed on total RNA extracted from testis (male symbol) or ovaries (female symbol) at various developmental stages as indicated in the Experimental Procedures section. The amplified cDNAs are indicated on the left margin. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control for the amount of cDNAs used in each PCR experiment. Right margin: Number of amplification cycles used in the PCR reaction. dpc, days postcoitum; dpp, days postpartum; Ad, adult; M, 1-kb DNA ladder molecular weight marker (GibcoBRL).

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Goat PRND was found to be expressed in both tissues, at all the developmental stages studied. This result is consistent with previously reported data obtained in bovine (Rondena et al., 2005) but differs from the expression pattern described in sheep, in which no expression was observed in prepubertal testis (Espenes et al., 2006).

PRND Expression Is Sex-Dimorphic in Goat Gonads

By semiquantitative RT-PCR, the PRND expression level was found to be constant in developing ovaries (Fig. 2). However, its level of expression consistently starts to increase in testes between 36 and 46 days postcoitum (dpc) stages. Thereafter and until adulthood, PRND expression remained sex-dimorphic, with higher levels in testes than in ovaries (Fig. 2). The 36 dpc stage corresponds to the beginning of testicular formation in he-goats (Pailhoux et al., 2002), and expression of various genes involved in sex-differentiation were found to be sexually dimorphic from this crucial developmental point (Pailhoux et al., 2005; Pannetier et al., 2006). It would thus suggest that, at least in goat, Doppel might be involved in testis differentiation. To further assess this potential role, the cellular and subcellular localization of this protein in developing gonads was investigated.

Cellular and Subcellular Localization of Doppel Protein in Goat Developing Gonads Parallels that of c-Kit

To determine the cellular and subcellular localization of Doppel protein in goat gonads, two previously reported anti-Doppel primary antibodies, directed against the ovine (Espenes et al., 2006) or bovine (Rondena et al., 2005) proteins, were tested. Both antibodies gave the same results, but stronger signals were obtained with the bovine anti-Doppel antibody (boDpl67-81). To precisely determine the cellular type expressing Doppel, two other antibodies were also used: one directed against AMH (anti-Müllerian hormone), a Sertoli-specific marker at these stages (Pailhoux et al., 2002); the other directed against c-Kit, a tyrosine kinase receptor specifically expressed by germ cells of both sexes and also by Leydig cells in male (Besmer, 1991; Buehr et al., 1993; Rothschild et al., 2003).

In early developing testes (44 dpc), Doppel was found in the cytoplasm of Leydig cells located in the interstitial compartment and in the nucleus of germ cells located inside the seminiferous tubules (Fig. 3). The Doppel cellular staining resembles those obtained with c-Kit–specific antibody, except for the subcellular localization that is in the membrane for c-Kit (Fig. 3). The AMH antibody was used to stain Sertoli cells, thus delineating the seminiferous tubules. At this early 44 dpc stage, there is no Sertoli-specific staining for Doppel. In the female 44 dpc ovaries, a strong nuclear signal is detected in the germ cells mainly located in the cortical area. Although Doppel is a glycophosphatidylinositol-anchored glycoprotein, its nuclear localization in germ cells at some developmental stages is not so surprising because its structurally related protein PrP (Mo et al., 2001) also possesses nuclear localization signals (Jaegly et al., 1998; Gu et al., 2003).

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Figure 3. AMH (anti-Müllerian hormone), c-Kit, and Doppel immunodetection in 44 days postcoitum (dpc) goat gonads. ao: AMH (a–c), c-Kit (d–f and j–l) and Doppel (g–i and m–o) immunofluorescent detection was performed on 44 dpc testes (a–i) and ovaries (j–o). The fluorescent staining is presented alone (a,d,g,j,m) or with a 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) blue nuclear-specific counterstaining (b,e,h,k,n and c,f,i,l,o). b,c,e,f,h,I,k,l,n: The third column (c,f,i,l,o) corresponds to a five times enlargement of the red rectangles depicted on the second column (b,e,h,k,n). c,f,i: To have a better view, some seminiferous tubules were delineated with a red trait. Some cells are marked as followed: Sertoli (yellow arrows), Leydig (white lozenge), and germinal (red arrowhead). At this stage, Doppel expression is detectable in the same cellular type as those expressing c-Kit both in male (Leydig and germ cells) and in female (germ cells) gonads. The Sertoli cells are highly positive only for AMH. Doppel staining is cytosolic in Leydig cells and nuclear in germ cells. Scale bars = 200 μm.

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In testes at the latter 62 dpc stage, Doppel continues to be mainly expressed in the cytosolic compartment of Leydig cells (Fig. 4). However, a fainter signal is also detectable inside the seminiferous cords in which both germ cells and Sertoli cells appear to be stained (Fig. 4). For Sertoli cells, the staining is clearly cytosolic, whereas for germ cells it can be in both compartments. In the 62 dpc female counterparts, only the germ cells were found positive, as for the 44 dpc stage. But at this latter stage, the signal remains nuclear only in a small population of germ cell lying just below the celomic epithelium. Most of meiotic germ cells, which are organized in ovigerous nests in the deeper cortex, were found positive for Doppel in their cytosol (Fig. 4). The cellular and subcellular localizations of Doppel appear thus to evolve with the differentiation stage of the male and female gonads (Table 1). The germinal cytoplasmic labeling seen at 62 dpc correlates that observed in other ruminants (Rondena et al., 2005; Espenes et al., 2006).

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Figure 4. AMH (anti-Müllerian hormone), c-Kit, and Doppel immunodetection in 62 days postcoitum (dpc) goat gonads. ao: AMH (a–c), c-Kit (d–f and j–l), and Doppel (g–i and m–o) immunofluorescent detection was performed on 62 dpc testes (a–i) and ovaries (j–o). The fluorescent staining is presented alone (a,d,g,j,m) or with a 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) blue nuclear-specific counterstaining ((b,e,h,k,n) and (c,f,i,l,o)). b,c,e,f,h,i,j,k,n,o: The third column (c,f,i,l,o) correspond to a 5 enlargement of the red rectangles depicted on the second column (b,e,h,k,n). To have a better view, some seminiferous tubules were delineated with a red trait (c,f,i). Some cells are marked as followed: Sertoli (yellow arrows), Leydig (white lozenge), and germinal (red arrowhead). At this stage, Doppel expression is quite similar to that of c-Kit in both sexes (Leydig and germ cells), but in contrast to c-Kit, Doppel is faintly detected in Sertoli cells. Doppel staining is cytosolic in all cellular types, except for a small population of female germ cells located just below the celomic epithelium, showing a nuclear signal. Scale bars = 200 μm.

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Table 1. Summary of the Cellular and Subcellular Localization of Doppela
 44 dpc62 dpc
CytosolNucleusCytosolNucleus
  • a

    dpc, days postcoitum.

TestisSertoli+
 Leydig++++++
 Germinal++++
OvarySomatic
 Germinal++++++

Although the subcellular localization seems to be different, this Doppel expression pattern appears quite similar to that observed for c-Kit, a protein known to be involved in gonad differentiation (for reviews, see Rossi et al., 2000; Choi and Rajkovic, 2006). Altogether, our observation is consistent with a role of Doppel in gonad differentiation in ruminants. This hypothetical newly identified function, apparently different from that of Doppel in mice (Behrens et al., 2002; Paisley et al., 2004), remains to be experimentally validated.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Animals and Tissue Samples

Procedures for handling animals were conducted in compliance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching (authorization no. 78-34). All goat fetuses were obtained from horned pregnant females, following hormonal treatments as previously described (Pailhoux et al., 2002). Day 0 postcoitum corresponded to the day of mating. The genetic sex of all fetuses was determined by PCR amplification of SRY and ZFY/ZFX genes, on liver genomic DNA (Pailhoux et al., 2002). For each fetus, one gonad was frozen in liquid nitrogen for molecular analysis; the other one was fixed for immunohistological studies.

Prnt Genomic Amplification and Sequence Analysis

Based on the recently published bovine genomic sequence (GenBank gi:78771396), primers were designed and used to amplify the PRNT coding region by PCR on bovine and goat genomic DNA. The sequence of the primers used and PCR conditions are given in Table 2. The amplified fragments were gel-purified and sequenced on both strands.

Table 2. Primers and PCR Conditionsa
Primer namesPrimer sequencesTemp./MgCl2
  • a

    PCR, polymerase chain reaction.

PRNP15′-AACCGCTATCCACCTCAGGG-3′56°C/ 1.5 mM
PRNP25′-AAAGAGGATCACACTTGC-3′ 
PRNT15′-CCCATTCATCTTTTCAGACT-3′50°C/ 1.5 mM
PRNT25′-CCAGAGCAGAAGAGAGATGGC-3′ 
PRND15′-ATGAGGAAACATCTGGGTGG-3′55°C/ 1.5 mM
PRND25′-AAGTCCTGCTCCCCTTTCCAACC-3′ 
GAPDH15′-AGGCCATCACCATCTTCCAG-3′58°C/ 1.5 mM
GAPDH25′-GGCGTTGGACAGTGGTCATAA-3′ 

RT-PCR

RNA extraction, DNase treatment, and cDNA synthesis were conducted as previously described (Pailhoux et al., 2001). Briefly, 2 μl (corresponding to 0.5 μg of reverse transcribed total RNA) of each RT mix was amplified in 50 μl of PCR reaction by using 0.5 U of Taq polymerase (TaKaRa), 200 μM of each dNTP, and 150 nM of each primer. The sequence of the primers used and PCR conditions are given in Table 2. All sets of primers were first validated on goat genomic DNA and were found to share similar efficiencies (data not shown).

Immunostaining

Freshly dissected gonads were fixed 1 hr in 4% paraformaldehyde in phosphate saline buffer (PBS) at 4°C. After washes in PBS with increasing concentrations of sucrose (0, 12%, 15%, and 18%), tissue specimens were embedded in Jung Tissue Freezing Medium (Leica Instruments) and frozen at −80°C. Cryosections (7 μm thick) were obtained and stored at −80°C. After thawing, sections were washed 10 min in PBS, then saturated with 5% bovine serum albumin and donkey serum 1/50 in PBS. After 3 washes in PBS (5 min each), the primary antibodies c-Kit (1/100, Santa Cruz Biotechnology), doppel boDpl67-81 (1/800, kind gift of Dr. Paltrinieri; Rondena et al., 2005), or AMH (1/50; Vigier et al., 1985) were applied onto the tissue sections overnight at 4°C. Negative controls were treated with a rabbit nonimmune serum (data not shown). After 3 washes, sections were incubated with fluorescein isothiocyanate–conjugated anti-rabbit secondary antibody (1/200, Vector) for 45 min at room temperature and rinsed 3 times in PBS. Slides were permanently mounted in Vectashield mounting medium with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI, Vector). Finally, images were acquired on an inverted Olympus IX 71 microscope equipped with a dedicated Cell-R imaging station (Olympus BioSystems - OBS) and an Orca ER Hamamatsu CCD camera.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The authors thank Patrice Congar for his help on microscopy observation. We also thank Dr. Saverio Paltrinieri (University of Milan, Italy) and Dr. Michael A. Tranulis (Norwegian School of Veterinary Science, Oslo, Norway) for providing anti-Doppel antibodies. A.K. is the recipient of a fellowship from the French Ministry of Research and Education. M.G. and I.P. are recipients of a Marie Curie Early Stage Research Training Fellowship of the European Community's Sixth Framework Programme.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES